Haynes Lab:Notebook/Engineering PC-TFs/2012/11/06: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(8 intermediate revisions by 2 users not shown)
Line 8: Line 8:
==Summary==
==Summary==
* <u>Gibson Assembly </u>
* <u>Gibson Assembly </u>
* Check with PCR


Assembly 1 contains PCR rxns 1,2, and 3. Assembly 2 contains PCR rxns 4,5.
Assembly 1 contains PCR rxns 1,2, and 3. Assembly 2 contains PCR rxns 4,5.
There is no addition of vector this time.
There is no addition of vector this time.
{|border="1" cellpadding="5" cellspacing="0" align="left"
{|border="1" cellpadding="5" cellspacing="0"  
|-
|-
! scope="col" style="background:#efefef;" |  
! style="background:#efefef;" |  
! scope="col" style="background:#efefef;" | 1
! style="background:#efefef;" | 1
! scope="col" style="background:#efefef;" | 2
! style="background:#efefef;" | 2


|-
|-
Line 27: Line 28:
|}
|}


<br>
<br>
<br>
<br>
<br>
<br>




{|border="1" cellpadding="5" cellspacing="0" align="left"
 
{|border="1" cellpadding="5" cellspacing="0"  
|-
|-
! scope="col" style="background:#efefef;" | <i>DNA fragments</i>
! scope="col" style="background:#efefef;" | <i>DNA fragments</i>
Line 71: Line 67:
|5.0 μL
|5.0 μL
|}
|}
<br><br><br><br>
----
'''PCR Check'''
# Product 1: outer-most primers for whole assembly
# Product 1: Gibson primer pair for hPCD
# Product 2: outer-most primers
# Product 2: Gibson primer pair for hPCD
<br>
<br>
{|border="1" cellpadding="5" cellspacing="0"
|-
| <i>Reagent</i> || 1 ||  2 || 3 || 4
|-
| Gibson product || 0.5 μL || 0.5 || 0.5 || 0.5
|-
| primer 1 || 1.0 || 1.0 || 1.0 || 1.0
|-
| primer 2  || 1.0 || 1.0 || 1.0 || 1.0
|-
| 2x GoTaq PCR mix || 14.5 || 14.5 || 14.5 || 14.5
|-
| dH<sub>2</sub>O || 12.0 || 12.0 || 12.0 || 12.0
|}
<br>
<br>
<br>
<br>
<br>
 
Program:
* 95°C, 3 min.
* [95°C, 30 sec; 57°C, 30 sec; 72°C, 30 sec] 30 cycles
* 72°C, 3 min.
* 4°C ∞


<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Revision as of 16:55, 6 November 2012

Engineering PC-TFs <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Summary

  • Gibson Assembly
  • Check with PCR

Assembly 1 contains PCR rxns 1,2, and 3. Assembly 2 contains PCR rxns 4,5. There is no addition of vector this time.

1 2
Gibson Rxn mix 15 μL 15 μL
DNA fragments 5 μL 5 μL



DNA fragments 1 2
#1 1.0 μL
#2 1.0 μL
#3 1.0 μL
#4 1.0 μL
#5 1.0 μL
dH2O 2.0 μL 3.0 μL
Total 5.0 μL 5.0 μL





PCR Check

  1. Product 1: outer-most primers for whole assembly
  2. Product 1: Gibson primer pair for hPCD
  3. Product 2: outer-most primers
  4. Product 2: Gibson primer pair for hPCD


Reagent 1 2 3 4
Gibson product 0.5 μL 0.5 0.5 0.5
primer 1 1.0 1.0 1.0 1.0
primer 2 1.0 1.0 1.0 1.0
2x GoTaq PCR mix 14.5 14.5 14.5 14.5
dH2O 12.0 12.0 12.0 12.0



Program:

  • 95°C, 3 min.
  • [95°C, 30 sec; 57°C, 30 sec; 72°C, 30 sec] 30 cycles
  • 72°C, 3 min.
  • 4°C ∞


Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/Engineering_PC-TFs/2012/11/06&oldid=651547"